The diseases caused by dengue viruses (DENV 1-4), mosquito-borne members of the family Flaviviridae, have emerged as the most important arthropod-borne viral diseases of humans. The frequency, geographical distribution, and severity of DENV epidemics have increased dramatically over the past decade. DENV is spread by mosquito species that also harbor Zika virus (ZIKV) and yellow fever virus. Although these viruses are endemic in tropical countries, they have also been reported in the southern states of the USA and are likely to move northward as global climate change increases their range. About half of the world's population is at risk for DENV infection including populations in the United States. For example, an outbreak of DENV in Hawaii from 2015 to 2016 involved 264 reported cases. Over 50 million people globally are estimated to suffer from the symptoms of dengue fever each year and of these at least 250,000 develop more severe symptoms with a fatality rate that can reach 20%. A repeat infection has a higher probability of being fatal. Early symptoms of DENV infection are similar to symptoms caused by many other hemorrhagic viruses including ZIKV, Ebola and yellow fever. Current serology cannot distinguish ZIKV and DENV infections. The lack of discriminating serology and the similar clinical presentations make it more difficult to rapidly identify the virus causing the symptoms, which delays effective patient treatment. This is a serious problem in areas where DENV and ZIKV are endemic and it is necessary to know which virus has infected a pregnant woman. Nucleic acid aptamers function as antibodies with tight and specific recognition of their targets, but have many advantages over antibodies, particularly for diagnostics. Advantages include stability to high temperatures (facilitating their storage and reusability), chemical synthesis that limits batch variation, and structural changes on binding ligand that enable their integration into many sensor platforms that are capable of rapid responses. Aptamers also prefer to interact with regions on a protein target than antibodies. The goal of this proposal is to develop an aptasensor with nucleic acid aptamers that can distinguish the NS1 proteins of DENV and ZIKV. The aptamers will be linked to nanoporous anodic aluminum oxide (NAAO) for detecting DENV infections. The following specific aims are proposed: 1) Select and mature a DNA aptamer that binds DENV NS1 with high affinity and specificity and test current ZIKV aptamers for specificity to ZIKV NS1 over DENV NS1, 2) Integrate the aptamers into an NAAO sensor and demonstrate the ability to distinguish DENV and ZIKV NS1 in the presence of serum proteins. With successful completion of these aims, the sensor will be optimized for specificity, signal/noise and accuracy in a phase II project to develop it for clinical application. Our ultimate goal is to develop a multiplexed sensor with aptamers to many hemorrhagic viruses that can be used by clinicians can obtain a rapid readout of the virus responsible for the presented symptoms to enable speedy initiation of the optimal treatment program.